This invention relates to a polymer brush that features polymer layers on a substrate surface. More specifically, the present invention is directed to a substrate having a first layer composed of a number of hydrophobic polymer chains attached to the substrate surface, and a second layer composed of a number of polymer chains, each of which include a water-soluble or water-dispersible segment having two termini, one terminus being free and the other being bound to a hydrophobic polymer chain. The present invention is particularly well suited for use as a sensor, wherein probes for biological molecules are attached to the water-soluble or water-dispersible segments. Sensors of this type are particularly useful for analyzing aqueous samples that contain biological materials. The present invention is further directed to methods of synthesizing such sensors, including a method wherein (i) an iniferter initiator is attached to the substrate surface in order to initiate polymer growth therefrom, and/or (ii) an activated monomer is used which yields polymer chains having functional groups to which probes can be attached without the need of a separate activation step.
Sensors for analyzing biological samples typically have the ability to process samples accurately and rapidly in an aqueous environment. This, in turn, looks to the presence of multiple probes on a single substrate surface capable of selectively interacting with components of the sample. For example, nucleic acid hybridization assays use multiple oligonucleotide probes bound to the substrate surface at pre-selected sites. The oligonucleotide probes, in turn, are available to participate in a hybridization reaction with selected nucleic acid components of the sample. Generally, this interaction of probe and sample relates to the utility of the components of the biological sample, such as the identity, concentration, purity or form of the components being sensed. There are generally many types of probes known, for example, antibodies that may immunoreact with a desired protein in a diagnostic assay, other protein binding assays, and dyes that change color to indicate the concentration of a desired protein, enzyme, small organic molecule, or inorganic molecule such as calcium or lithium.
Attaching probe molecules to surfaces is typically difficult for a number of reasons. For example, the surfaces often lack functional groups that are uniquely reactive in an aqueous system, or that are readily accessible to the probe molecules (as a result of factors such as surface crowding or steric hindrance). The latter problem becomes particularly acute as the number of functional groups per unit area of surface increases. In addition, once probes are bound to the surface, they must remain accessible to components of the biological sample. Here, too, factors such as steric hindrance may hamper accessibility. Molecular crowding (i.e., density) becomes a critical issue as well, particularly in systems where fluorescence quenching can be an issue. Finally, given that the biological samples to be analyzed (or “probed”) are often aqueous, hydrolysis of the bond or linkage which holds the probe to the substrate surface can result in detachment of the probe, thus reducing signal sensativity.
Controlled free radical polymerization methods with living-type kinetics have been used to covalently bond polymers to the surfaces of substrates and thereby form “polymer brushes.” Husseman et al., Macromolecules 1999, 32, 1424–31, for example, describe a variety of polymer brushes prepared using such methods. The resulting brushes, however, were not water-soluble or water-dispersible and thus were not suitable for applications involving aqueous samples such as biological samples. As a result, the stability of bonds linking the polymers to the substrate surface, when exposed to an aqueous environment, was not addressed. Additionally, Husseman et al. failed to address the importance of controlling the grafting density of, or spacing between, the polymer chains attached to the substrate surface, in order to optimize both the number of probes which may be attached for a given application, as well the efficiency of those probes, once attached, to interact with the target molecules.
Accordingly, a need continues to exist for a polymer brush, as well as a process for the preparation thereof, wherein the polymer chains are stably bound to the substrate surface, such that the polymer chains will not detach when used in an aqueous environment. Such polymer chains will preferably have a controlled molecular architecture (i.e., composition, functionality, molecular weight, polydispersity, etc.), as well as spacing or grafting density, such that the attachment of probe molecules of a given size or type can be optimized. Such brushes would thus enable the preparation of a sensor (i.e., a polymer brush having probe molecules attached thereto) having enhanced stability in an aqueous environment and a controlled structure, such that probe accessibility to the target biological molecules can be optimized.